Objective: We knocked out the genes related to lipopolysaccharide in outer membrane of Escherichia coli BL21 (DE3) to study the effects on extracellular secretion of recombinant proteins.
Methods: We generated waaF or msbB knockout mutants [E. coli BL21 (ΔwaaF) or E. coli BL21 (ΔmsbB) ] of E. coli BL21 (DE3) by using lambda-Red recombination system. Then, we transformed recombinant plasmids pET-ffase or pET-pga into E. coli BL21 (AmsbB) , E. coli BL21 (ΔwaaF) and E. coli BL21 (DE3) respectively, to generate the engineering strains E. coli BL21 (ΔmsbB)/ pET-ffase, E. coli BL21 (ΔwaaF)/pET-ffase, E. coli BL21 (DE3)/pET-ffase, E. coli BL21 (ΔmsbB)/pET-pga, E. coli BL21 (ΔwaaF)/pET-pga and E. coli BL21 (DE3)/pET-pga. Finally, we studied the effects of mutants on extracellular secretion of beta-fructofuranosidase (EC 3. 2. 1. 26, beta-FFase) and penicillin G acylase (EC 3. 5. 1. 11) in shaking flask fermentation.
Results: After induced expression for 4 hours, up to 19.7% of the beta-FFase activity was found in the culture medium with the msbB deletion mutant, and 50.9% with the waaF deletion mutant, compared to the original 2.6%. Besides, after induced expression for 24 hours, up to 1708 U/L extracellular activity of penicillin G acylase was found in the culture medium with the waaF deletion mutant, which was 4.1 times of the original.
Conclusion: Knockout mutants (ΔmsbB and ΔwaaF) had significantly higher excretion of beta-FFase and the waaF deletion mutant had higher excretion of penicillin G acylase.